Abstract
Acute myeloid leukemia (AML) exhibits a hierarchical cellular organisation, with a minor fraction of self-renewing leukemic stem cells (LSCs) at the apex of this hierarchy. LSCs confer resistance to chemotherapy and are implicated in AML relapse. Compared to normal haematopoietic stem cells, upregulated CD123 or IL-3Rα was one of the first cell surface markers identified on LSCs and is particularly highly expressed in FLT3-ITD and RUNX1 mutated AML. Thus, identifying the molecular mechanisms responsible for upregulating CD123 could offer a promising avenue for the eradication of LSCs. Here we illustrate a novel role for TGF-β in upregulating CD123 and promoting quiescence and chemotherapy resistance.
Method We performed flow cytometry, qPCR and western blot analysis of cytokine stimulations and inhibitor studies with ligand-trap, TGF-β receptor 1 (TβRI) inhibitor, gilteritinib and midostaurin in AML cell lines and primary samples, and CD34⁺ cord blood (CB) stem cells. We did SMAD2/4 co-immunoprecipitation, chromatin immunoprecipitation, immunofluorescence and shRNA knockdown studies in the TF-1 and MOLM13 cells. We performed CFU assays for TGF-β-treated CD34⁺, TβRI inhibitor–treated primary AML samples and cytarabine/TGF-β-treated TF-1 cells. We did propidium iodide cell cycle and cytarabine-induced apoptosis assessment via AnnexinV/7AAD.
ResultWe first identified TGF-β1 mediates upregulation of IL-3Rα by 4 to 5-fold increase on the cell surface and total protein, as well as corresponding IL3RA RNA (6-fold increase) in the TF-1 cells. Similarly, healthy common myeloid progenitors (CMPs) (CD34+CD38+CD45RA-CD123low) from CB exhibited a 2-fold increase in IL-3Rα expression following TGF-β1 treatment. Focusing on TGF-β family members, we found that only TGF-β1 and TGF-β2, but not BMP4, through TβRI/TβRII activation could upregulate IL-3Rα expression. TGF-β1 binding to TβRI/TβRII results in consistent SMAD2 phosphorylation at its C-terminal region, forming a complex with SMAD4. Using chromatin immunoprecipitation, we detected SMAD2 and SMAD4 directly bound to the IL3RA proximal enhancer region upon TGF-β1 stimulation. We further confirmed that SMAD4 knockdown blocked the ability of TGF-β1 to upregulate IL-3Rα.Mechanistically, we found that IL-3 exerted a positive feedback effect on the TGF-β1 signalling pathway by upregulating the expression of TβRI, leading to enhanced phosphorylation of SMAD2. This, in turn, resulted in a further increase in expression of the IL-3Rα, suggesting a reinforcing loop between IL-3 and TGF-β1 signalling components. Both TGF-β1 alone and in combination with IL-3 induced cellular quiescence, as evidenced by a ~30% increase in the proportion of cells in the G0/G1 phase and a corresponding decrease in the S and G2/M phases of the cell cycle. This quiescent state was accompanied by elevated expression of the cyclin-dependent kinase inhibitor CDKN1A (p21), further supporting the role of TGF-β1 in cell cycle arrest. Functionally, TGF-β1 conferred a survival advantage following cytarabine treatment. While cytarabine alone induced significant apoptosis, detected by AnnexinV and 7AAD, TGF-β1 co-treatment reduced cell death by approximately 50%. This protective effect was corroborated by colony-forming unit (CFU) assays, which showed a 20-fold increase in colony numbers in cells treated with both TGF-β1 and cytarabine compared to cytarabine treatment alone.We then investigated leukemias harboring FLT3-ITD and RUNX1 mutations, which have high CD123 expression. We found FLT3-ITD mutations hijack the TGF-β1 pathway by inducing constitutive phosphorylation of SMAD2 at its linker region, sensitive to gilteritinib and midostaurin. This aberrant signalling is associated with increased expression of TGF-β1-responsive genes, including TGFB1, SMAD2, CDKN1A, and IL3RA. Primary RUNX1 mutated patient sample with high CD123 expression LSCs population (CD34+CD38- CD45low) were sensitive to TβRI inhibition (≅2-fold decrease) and their colony formation ability decreased by 50% when they were pretreated with TβRI inhibitor.
ConclusionGiven the well-established role of LSCs with high expression of CD123 in post-chemotherapy relapse and the frequent co-occurrence of FLT3-ITD and RUNX1 mutations in AML patients, our study underscores the clinical relevance of targeting the TGF-β1 pathway. These findings highlight TGF-β1 signalling as a key modulator of LSC persistence and a potential therapeutic vulnerability in AML.
